RNA interference (RNAi) is emerging as one of the most powerful technologies for sequence-specific suppression of genes and has potential applications ranging from functional gene analysis to therapeutics. Due to the relatively low immunogenic and oncologic effects, the development of non-viral delivery methods in vitro and in organisms is of considerable current interest. In recent years, a number of strategies have been developed based on liposomes, gold and silica nanoparticles (NPs), cationic and biodegradable polymers, and peptides. The delivery efficiency, however, remains low, especially under in vivo conditions. Another limitation shared by all the existing delivery technologies is the lack of an intrinsic signal for long term and real-time imaging of siRNA transport and release. Such imaging could provide important information on rational design of siRNA carriers. Currently, organic fluorophores are used to label siRNA or the delivery vehicles. However, the photobleaching problem associated with essentially all organic dyes prevents long-term tracking of siRNA-carrier complexes. Similarly, electron-dense gold NPs are visible under transmission electron microscope (TEM) and provide the highest imaging resolution in fixed cells, but they do not allow real-time imaging of live cells.
QDs have been used for siRNA delivery and imaging. However, these QD probes are either mixed with conventional siRNA delivery agents (Lipofectamine™) or external endosomal rupture compounds (e.g., chloroquine) for gene silencing activity, significantly limiting their potential applications in vivo. Therefore, development of multifunctional QDs with integrated functionalities of cell binding and internalization, endosome escape, siRNA protection against enzyme activities, siRNA unpackaging (siRNA-carrier dissociation), and siRNA tracking is of urgent need. Furthermore, packaging these functionalities into single nanoparticles also represents a significant technological challenge.
In one aspect, the present invention seeks to fulfill this need and provides further related advantages.
Combination of gene vectors such as liposome, cationic polymers and recombinant viruses with magnetic nanoparticles or microparticles allows rapid delivery of DNAs and RNAs into cells, a process also referred to as magnetofection. In addition to the magnetic force directed delivery, another key feature of magnetofection is that it is capable of reaching similar transfection efficiency at significantly reduced DNA and RNA concentrations. Protocols on how to transfect nucleic acids to both suspended and adherent cells (including primary neurons) using cationic liposomes and polymers associated with MNPs has been recently reported. Similarly, the combination of magnetic microspheres of various sizes with recombinant adeno-associated viral vectors for increased gene transduction efficiency and modified in vivo biodistribution has also been recently reported.
Despite these recent advances, a major drawback of the magnetofection carriers is that the fully assembled magnetic vectors are based on large aggregates of MNPs, nucleic acids, and cationic lyposomes (or polymers), often in the sub-μm to μm range. The aggregated MNPs are highly responsive to external magnetic fields compared with the original single MNPs. However, aggregated MNPs are associated with problems related to their size. That is, aggregated MNPs cannot be further developed into biomolecularly targeted and MRI traceable drug delivery vehicles because it is extremely difficult to control the aggregation process for compact and uniform nanoparticle clusters. This is indeed evidenced by the absence of magnetofection of siRNA in vivo, since the first reported data on siRNA magnetofection in 2003. Instead, the rational design of dispersed nanostructures with precisely tunable sizes, integrated targeting, imaging, and therapeutic functionalities, has become the most promising route for efficient siRNA delivery.
Recent advances in high-temperature non-hydrolytic nanoparticle synthesis has led to the development of highly monodisperse MNPs with size tunability ranging from a few nanometers to approximately 50 nm in diameter, which is suitable to many biomedical applications. For example, it has been demonstrated that magnetic resonance signals from MNPs of 4 to 12 nm vary drastically. Using the same MNP size range, MNPs response to magnetic fields have been shown to be highly size dependent, which opens new opportunities for simultaneous separation of complex mixtures. Similarly, in nanoparticle based siRNA delivery, the particle size is also one of the most important factors in that it affects the particle diffusion, in vivo biodistribution, plasma circulation time, and surface functionalities (e.g., curvature and number of ligands). Uniform MNPs can be routinely made with nanometer precision, which is difficult, if not impossible, to achieve with traditional aqueous-based synthetic approaches (used to make MNPs in essentially all the reported magnetotransfection studies). However, until now these monodisperse MNPs have been mainly used in bio-separation and in vivo MRI.
In another aspect, the present invention seeks to fulfill this need and provides further related advantages.